[0001] This invention was discovered in the performance of U.S.govemment supported research
under grants NIMH KO2MH01366 and NCEMS GM52213, and the U.S. government may have certain
rights in the invention.
DESCRIPTION
BACKGROUND OF THE INVENTION
Field of the invention
[0002] The present invention relates to the synthesis of novel ligands selective for a subgroup
of receptors for serotonin (5-HT). While there are seven subgroups of 5-HT receptors,
this invention is selective for the 5-HT
6 subgroup. This invention also relates to the synthesis of novel ligands selective
for the 5-HT
6 subgroup receptor that act as agonists to the natural ligands for this receptor.
The invention also relates to the creation of novel ligands that act as antagonists
to the 5-HT
6 receptor. The invention further relates to use of said compounds to treat mammals
adversely affected by conditions mediated by the 5-HT
6 receptor.
Description of the prior art
[0003] Serotonin receptors have been divided into a number of families and subfamilies (5-HT
1-5-HT
7) and approximately 14 populations have been cloned. One of the newest populations
identified is the 5-HT
6 subgroup. It has been observed that various tricyclic psychotropic agents (neuroleptics,
atltidepressants, and atypical neuroleptics agents) bind the 5-HT
6 receptor with nanomolar affinities (Roth et al. J. Pharmacol. Exp. Ther. 1994, 268,
1403-1410). A rat 5-HT
6 receptor was first cloned in 1993 and, more recently, the same group described the
cloning of a human 5-HT
6 receptor. The 5-HT
6 serotonin receptors are members of the G-protein superfamily, are positively coupled
to an adenylate cyclase second messenger system, and are found primarily in the central
nervous system. Serotonin bound to the 5-HT
6 receptor subgroup causes an activation of the adenylate cyclase enzyme, with concomitant
increased levels of intracellular cAMP. Although the exact physiological function
and clinical significance of the 5-HT
6 receptor subgroup is not known, as noted above, many anti- psychotic agents bind
these receptors with high affinity. Also, in rats that do not express 5-HT
6 receptors, the animals behave in a manner that seems to involve an increase in cholinergic
function, suggesting that 5-HT
6 specific ligands might be of value in the treatment of anxiety-related disorders
and memory deficits.
[0004] Upon binding to cellular receptors, ligands may act as agonists or antagonists to
endogenous receptor-ligand function. In the case of the 5-HT
6 receptor, several specific ligands have been discovered which act as 5-HT
6 specific antagonists, but prior to the present invention, selective ligands which
act as agonists to the 5-HT
6 receptor were unknown.
SUMMARY OF THE INVENTION
[0005] It is an object of the invention to create derivatives of serotonin (5-HT) that specifically
bind the 5-HT
6 receptor subgroup of the serotonin receptor family. It is another object of this
invention to create 5-HT
6- selective ligands that act as agonists when bound to the 5-HT
6 receptor. It is further an object of this invention to create 5-HT
6-selective ligands that act as antagonists when bound to the 5-HT
6 receptor. Furthermore, the compounds of the present invention that possess antagonist
activity are tryptamine derivatives and are structurally unrelated to previously described
5-HT
6 antagonists. It is further an object of this invention to administer 5-HT
6 selective ligands to animals to determine the physiological and biochemical effects
of specific activation and inhibition of 5-HT
6 receptor function. Finally, it is an object of this invention to treat mental disorders
mediated by 5-HT
6 function by administering to treatment subjects the 5-HT
6-selective agonists and antagonist compounds described herein.
[0006] Various indolealkylamines, including serotonin(5-HT) and 5-methoxytryptamine, have
been observed to bind the 5-HT
6 receptor with high affinity and produce a potent dose-dependent increase in cAMP
levels. These tryptamines, however, are non-selective and bind at multiple families
of 5-HT receptors. According to the invention, various modifications of 5-HT have
been made to generate ligands with selectivity for the 5-HT
6 receptor. An analog of 5-HT with a 2-methyl substituent introduced (2-methyly-5-HT)
binds the 5-HT
6 receptor with an affinity equivalent to that of the parent compound. The above analog
is selective for the 5-HT
6 and 5-HT
3 receptors and binds at the 5-HT
6 subgroup with a 20 fold greater affinity than at 5-HT
3 receptors.
[0007] A 2-methyl analog of 5-methoxytryptamine, 5-methoxy-2-methyltryptamine, binds to
the 5-HT
6 receptor with an affinity comparable to 2-methyl-5-HT. However, 5-methoxy-2-methyltryptamine
lacks affinity for 5-HT
3 receptors. Thus, 5-methoxy -2-methyltryptamine presents a ligand with specificity
for the 5-HT
6 receptor subgroup. In the present invention, the 5-methoxy-2-methyltryptamine compound
has been modified and several of its alkyl derivatives bind with comparable affinity
and activate adenylate cyclase activity at levels comparable to serotonin. Furthermore,
one derivative, 5-methoxy-2- phenylotryptamine, binds to the 5-HT
6 receptor with a high affinity but the phenyl addition renders the compound an antagonist
to 5-HT stimulated adenylate cyclase activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] The foregoing and other objects, aspects and advantages will be better understood
from the following detailed description of the preferred embodiments of the invention
with reference to the drawings, in which:
Figure 1 is a graph showing the adenylate cyclase activity observed with several compounds
of the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
[0009] The invention provides novel tryptamine derivative compounds with selectivity for
the 5-HT
6 receptor subgroup. For the present purpose, an agent is termed selective when it
displays an affinity for 5-HT
6 receptors that is tenfold higher than affinities it displays for other related receptor
populations. The invention further provides a method using said compounds to receptor
subtype 5-HT
6 as agonists, or as antagonists to serotonin. The compounds of the invention can be
used either as the free base or as the pharmaceutically acceptable acid-addition salt
form, for example, hydrochloride, hydrobromide, tartrate, and maleate. They may be
used in oral or injectable pharmaceutical preparations as prophylactic and acute-phase
remedies for the relief and reversal of serotonin-regulated symptoms. They may be
used alone or in combination with each other or other known medications. Finally,
said compounds may be used as above for determining 5-HT
6 receptor function.
[0010] Serotonin (5-hydroxy tryptamine, or 5-HT) is a product of tryptophan metabolism and
is a tryptamine derivative that is a potent neurotransmitter. Serotonin is a well-characterized
tryptamine derivative which regulates calcium ion channels on the surface of nerve
and muscle cells. Many mental disorders in humans are associated with fluctuations
in serotonin levels and are effectively treated with drugs which specifically interact
with serotonin receptors or that block the reuptake of serotonin into the presynaptic
axon terminals, suggesting that serotonin dysregulation may be involved in various
mental disorders. Some serotonin receptor ligands and are clinically approved as drugs
for the treatment of migraine headaches, depression, high blood pressure, and psychosis.
[0011] Generally, tryptamine derivatives are non-selective and bind at multiple 5-HT receptor
subgroups. Serotonin is no exception and binds at the various subfamilies of the 5-HT
receptor, including the 5-HT
6 subgroup where it is a potent activator of adenylate cyclase enzyme activity. Serotonin
has the chemical formula:
[0012] Some modifications of serotonin result in loss of affinity for various 5-HT receptor
subgroups. It had previously been thought that introduction of a 2-methyl substituent
to 5-HT was not tolerated by any 5-HT receptors but the 5-HT
3 subgroup. Thus, prior to identification of the 5-HT
6 receptor, 2- methylation of 5-HT was thought to render the product selective for
the 5-HT
3 subgroup. We have found that the two methyl derivative of 5-HT, 2-methyl-5-HT has
a high affinity for the 5-HT
6-receptor. In fact, 2-methyl-5-HT binds the 5-HT
6 receptor with a 20 fold greater affinity over 5-HT
3 receptors. The 5-HT
6-selective ligand 2-methyl-5-methoxytryptamine contains a primary amine, presenting
an obstacle to the compound crossing the blood brain barrier and also rendering the
compound vulnerable to rapid metabolism due to oxidative deamination. Our discovery
that a methyl substituent at the 2 position was tolerated by the 5-HT
6 receptor, together with the previous observation that O-° methylation of 5-HT abolishes
affinity for 5-HT, receptor, led to the present invention.
[0013] To address the above limitations of 2-methyl-5-methoxytryptamine, several derivative
compounds were synthesized that were lipophilic and also might be less prone to rapid
metabolism. N,N-dimethyl substituents were added to 2-methyltryptamine to create 2-methyl-N,N-dimethylayptamine
(Ki=308nM). Re-introduction of the methoxy group to this compound, to form 2-methyl-5-methoxy-N,N-dimethyltryptamine
resulted in a compound (Compound A) with an affinity for the 5-HT
6 receptor of Ki=60nM. Homologation of the 2-methyl substituent of the above compound
to form 2-ethyl-5-methoxy-N,N-dimethyltryptamine resulted in a ligand with an increased
affinity for the 5-HT
6 of Ki=16nM (Compound B). To determine whether or not greater bulk additions could
be added in place of a methyl or ethyl group, the 2-phenol derivative of the 2-methyl-5-methoxy-N,N-dimethyltryptamine
was generated. This compound D binds the 5-HT
6 receptor with a Ki=20nM. These derivatives were of the general formula 1
where R
1 and R
2=H or CH
3,
R
5=H, OH, OCH
3, or a substituted or unsubstituted alkyl,
R
4=H, CH
2-phenyl, SO
2-phenyl, or CH
2 as part of a substituted or unsubstituted alkyl ring connecting R
4 and R
5,
R
5=H, CH
3, or CH
2 as part of a substituted or unsubstituted alkyl ring connecting R
5 with either R
4 or R
6,
R
6=H, CH
3, or CH
2 as part of a substituted or unsubstituted alkyl ring connecting R
6 and R
5. Thus, the present invention relates to the compounds as defined in any one of claims
1 to 6 or 9.
[0014] The compounds of the invention and the pharmaceutically acceptable salts of the compounds
of the invention can be used in the form of pharmaceutical preparations. The preparations
can be administered orally, for example in the form of tablets, coated tablets, dragees,
hard and soft gelatin capsules, solutions, emulsions or suspensions. The administration
can be effected rectally, for example in the form of suppositories, or parenterally,
for example in the form of injection solutions.
[0015] The compounds of the invention can be processed with pharmaceutically inert, inorganic
or organic carriers for the production of pharmaceutical and research preparations.
The preparations can contain preservatives, solubilizers, wetting agents, emulsifiers,
sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers,
masking agents or antioxidants. They can also contain still other therapeutically
valuable substances.
[0016] The compounds of the present invention can also be radiolabelled and used to identify
other 5-HT
6 ligands using techniques common in the art. This can be achieved by incubating the
receptor in the presence of a ligand candidate plus an equimolar amount of radiolabelled
compound of the invention. Ligands selective for 5-HT
6 are then revealed as those that are not significantly displaced by the compounds
of the present invention.
[0017] Another embodiment of the invention can be the administration of the compounds of
the invention to animals in drug discrimination assays. In a drug discrimination paradigm,
animals (usually rats) can be trained to recognize the effects of a given agent. Once
trained, these animals can be used in tests of stimulus generalization to identify
other agents that produce similar effects (i.e., agonists), or the animals can be
used in tests of stimulus antagonism to identify agents that block or antagonize (i.e.,
antagonists) the effects of the training drug. Hence, the procedure can be used to
identify agonists that produce an effect common to the training drug, more antagonists
that can block the effects of the training drug. Specifically, with a 5-HT
6-selective agonist as training drug, the animal can be used to identify other 5-HT
6 agonists and to identify 5-HT
6-antagonists.
[0018] One family of compounds contemplated for use in this invention is represented by
the formula 2
wherein R
2 is selected from the group consisting of ethyl, n-propyl, and phenyl.
[0019] Another family of compounds for use in this invention is represented by formula 3
wherein R
1 is selected from the group consisting of lower alkyls such as ethyl and propyl, methyl
and hydrogen, and can be the same or different at each location, and R
4 is from the group comprising H, methyl, ethyl or propyl.
[0020] The following examples illustrate the present invention in more detail.
Example 1
[0021] Synthesis of 2-ethyl-5-methoxy-N,N-dimethyltryptamine maleate (Compound B). A 2.5M
solution of nBuLi (1.75 ml. 4.38 mmol) was added in a drop wise manner to a stirred
solution of 2-methyl-5-methoxy-N,N-dimethyltryptamine (compound A in the examples
below) free base (1.00 g, 4.33 mmol) in dry THF (7 mL) at -78 °C under N
2.
[0022] After stirring the reaction mixture for five minutes, the cooling bath was removed
and CO
2 gas was passed into the solution for 10 minutes. The solvent was removed at 0 °C
under reduced pressure to give a transparent solid. The flask was flushed with N
2 and dry THF (7 mL) was added. The reaction mixture was degassed at -150 °C under
reduced pressure of 1mMHg, then allowed to warm to -78 °C; 1.7M tNuLi (2.8 mL, 4.8
mmol) was added in a drop wise manner. The solution was kept at -78 °C for three hours.
The reaction make sure was acidified with a saturated ethereal solution of HCl. Anhydrous
Et
2O was added to the resulting suspension and the supernatant was decanted. The residue
was heated at 100 °C under reduced pressure for 20 minutes. The resulting residue
was purified by flash chromatography on silica gel (CH
2Cl
2/MeOH; 12:1) to give 0.17 g of a bright yellow oil (16%).
1H-NMR(CDCl
3) d 8.06 (s, 1H, J=8.67 HZ), 6.98 (s, 1H), 6.76 (dd, 1H, J=2.34, 8.73 HZ), 3.84 (s,
3H) 2.91-2.87 (m, 2H), 2.71 (q, 2H, J=7.38 HZ), 2.57-2.52 (m, 2H) 2.38 (s, 6HJ)1.25
(t, 3H, J=7.38 HZ).The maleate salt was prepared and recrystallized from an EtOAc/Et
2O mixture; mp 123 °C.
Example 2
[0023] Magnesium turnings and NH
4Cl were added to a solution of Example 11
infra (1-Benzenesulfonyl-5-methoxy-2-n-propyl-N,N-dimethyl tryptamine, free base; 259 mg,
0.65 mmol) in MeOH (17 mL) and the mixture was allowed to stir at room temperature
for one-hour. Saturated NH
4Cl solution was added and the reaction mixture was extracted with CH
2Cl
2. The organic portion was dried (MgSO
4) and the solvents was removed under reduced pressure. The residue was purified by
flash chromatography on silica gel (CH
2Cl
2/MeOH; 9: 1) to give 75 mg of a bright yellow oil.
1H-NMR (CDCl
3) 0 7.71 (brs, 1H), 2.89-2.83 (m, 2H), 2.69 (t, 2H, J=7.56 Hz), 2.53-2.47 (m, 2H),
2.36 (s, 6H), 1.68 (tq, 2H, J=7.28, 7.56 HZ), 0.98 (t, 3H,J=7.28 HZ). The salt was
prepared and recrystallized from acetone; mp 146-147 °C.
Example 3
[0024] 5-methoxy-2-pbenyl-N,N-dimethyltryptamine oxalate (Compound D). 5-methoxy-2-phenylindole
(3 g, 13.44 mmol) was added to a stirred ice-cooled solution of 1-dimethylamino-2-nitroethylene
(1.56 g, 13.44 mmol) in trifluoracetic acid (8ml). The resulting mixture was allowed
to stir under N
2 at room temperature for 30 minutes and was then poured into ice/water. The solution
was extracted with EtOAc and the organic portion was washed consecutively with saturated
NaHCO
3 solution, H
2O, and then brine. The organic portion was dried (MgSO
4) and solvent was removed under reduced pressure. The residue was recrystallized from
CH
2Cl
2/hexane to give 2.36 g (60%) of a red powder.
1H-NMR (acetone-d6) d 8.82 (brs, 1H), 3.92 (s, 3H), 1R (Kbr)1601, 1475, 1251 cm
-1. A solution of this material (2 g, 6.75 mmol) in dry THF was added in a drop wise
manner to a cooled 0 °C suspension of LiALH
4 (1.54 g, 40.5 mmol)in dry THF under N
2. The reaction mixture was heated at reflux for one-hour and then allowed to stand
at room temperature overnight. The resulting mixture was quenched with H
2O then 15% NaOH solution. Celite was added and the solution was filtered. The solvent
was removed under reduced pressure. The residue was purified by flash chromatography
on silica gel (CH
2Cl
2/MeOH; 9:1) to give 1 g (55 %) of the primary amine as an oil.
1H-NMR (CDCl
3) d 8.19 (brs, 1H, J=2.37 HZ), 6.88 (dd, 1H, J=2.24, 8.75 HZ), 3.89 (s, 3H), 3.04
(brs, 4H). IR(KBr) 3397, 3347 cm
-1. Sodium cyanoborohydride (510 mg, 8.12 mmol) was added to a solution of the primary
amine (700 mg, 2.63 mmol) and 37% aqueous CH
2O in MeCN (10 mL) at room temperature. The resulting mixture was adjusted to pH 5
with HOAc and was allowed to stir at room temperature overnight. A 15% solution of
NaOH was added to neutralize the mixture and the mixture was extracted with. CH
2Cl
2. The combined organic portion was washed with saturated NaHCO
3 solution and brine. The organic portion was dried (MgSO
4)and solvent was removed under reduced pressure. The residue was purified by flash
chromatography on silica gel (CH
2Cl
2/MeOH; 9: 1) to give 195 mg of 5-methoxy-2-phenyl-dimethyltryptamine free base as
a white powder.
1HNMR (CDCl
3) d 8.05 (brs, 1 h), 7.56-7.53 (m, 2H),7.49-7.44 (m, 2H), 7.39-7.34 (m, 1H), 7.29,
7.25 (m, 1H), 7.11 (d, 1H, J=2.25 HZ). 6.87 (dd, 1H, J=2.52, 8.73 HZ), 3.89 (s, 3H).
3.13-3.08 (m, 2H), 2.72-2.66 (m, 2H), 2.39 (s, 6H). Although the HCl salt has previously
been reported, difficulties in his purification led to isolation of the product has
its salt; after recrystallization from acetone.
Reference
Example 4
[0025] 4-dimethylaminomethyl-9-benzyl-6-methoxy-1,2,3,4-tetrahydrocarbazole hydrochloride
(Compound E). A mixture of 4-methoxyphenyl)benzylamine (42 g 0.2 mol) and ethyl 6-bromocyclohexanone
Carboxylate (J. Org Chem 1961, 26, 22) (24.9 g, 0.1 mol) were heated at reflux in
dry benzene (250 mL) for 24 hours. The reaction mixture was cooled and precipitated
(4-methoxyphenyl) benzylamine hydrobromide was separated by filtration. The benzene
extract was concentrated and fused zinc chloride (40 g) was added in reflux in absolute
ethanol (125 mL) for six hours. The cooled mixture was slurred in H
2O (250 mL) and extracted with (Et
2O; 4x 200 mL). The combined ether extracts were washed with 5% HCl (100 mL), followed
by brine solution and dried with MgSO
4. The ether extract was evaporated under reduced pressure to give crude ethylester
of title compound which was treated with a solution of KOH (50 g) in H
2O (150 mL) and CH
3OH (150 mL) at reflux temperature for three hours. The solution was evaporated to
dryness under reduced pressure, the resulting residue was dissolved in H
2O (250 mL) and the aqueous solution was extracted with Et
2O and acidified with 10 % HCl. The resulting solid was dried to give 22 g (33%) of
title compound and was recrystallized from ls.PrOH-H2). Mp 212-214 °C. To a mixture
of sodium hydride (0.96 g, 0.04 mol) in dry benzene (200 mL) was added portion wise
9-benzyl-6-methoxy-1,2,3,4-tetrahydro-4-carboxylic acid (14.00 g, 0.04 mol) and the
mixture was stirred for one-hour. Thionoyl chloride (3.00 mL, 0.04 mol) was added
slowly in the stirring was continued for 30 minutes. The resulting solution was poured
into aqueous dimethylamine solution (40 %)(36.50 mL) with ice bath cooling. The mixture
was third for one-hour, washed with 100 mL H
2O, NaHCO
3 (50 mL)and saturated brine solution (50 mL), and dried with MgSO
4, diluted with n-pentane (200 mL)and cooled to give 4-dimethylaminocarbonyl-9-benzyl-6-methoxy-1,2,3,4-tetrahydrocarbazole
(9.10 g, 60 %) mp 153-155 °C. To a stir it solution of LialH
4 (4.71 g, 94.2 mmol) in dry THF was added portion wise 4-dimethylaminocarbonyl-9-benzyl-6-methoxy-1,2,3,4-tetrahydrocarbazole
(9.00 g, 24.8 mmol) and the mixture was heated under reflux for five hours the reaction
mixture was cooled water (5.0 mL) NaOH solution (5.0 mL) was added and filtered. The
filtrate was evaporated to dryness to give 4-dimethylamino-9-benzyl-6-methoxy-1,2,3,4-tetrahydrocarbazole
(8g, 92 %). The free base was dissolved in either in converted to the salt using ethereal
hydrochloride and recrystallized from a mixture of EtOH and Et
2O, Mp 238-240 °C.
Reference
Example 5.
[0026] Compound F is known and was prepared according to the following patent procedure:4-aminomethyl-9-benzyl-1,2,3,4-tetrahydrocarbazoles,
U.S. patent No. 3,939,177. Feb 17, 1976.
Example 6
[0027] Compound G Sodium metal was added portion wise over a thirty minute period to a stirred
solution of 4-(dimethylaminomethyl)-9-benzyl-6-methoxy-1,2,3,4-tetrahydrocarbazole
(4 g, 0.01 mol) in liquid NH
3 (300 mL). NH
4Cl (3.0 g) was added until the blue-collar of the mixture dissipated. The NH
3 was evaporated, water (50 mL) was added in the mixture was extracted withCH
2CL
3. The combined organic portion was washed with water (50 mL), brine (50 mL), dried
(MgSO
4) and evaporated to give an oil. The oil was purified by column chromatography (CHCL
3/MeOH; 9:1) and converted to an oxalate salt. The salt was recrystallized from anhydrousEt
2O/absolute-EtOH to give 1.8 g of the desired target as a white powder.
1H-NMR (CDCl
3, free base) d 8.10 (s, 1H, NH), 7.20 (t, 1H, ArH), 6.90 (d, 1H, ArH), 6,70 (dd, 1H,
ArH), 3.80 (s, 3H, OCH3), 3.40 (t, 1H, CH), 3.15 (d, 1H, CH), 3.0 (t, 1H, CH)3.00
(t, 1H, Ch), 2.82 (s, 6H, 2XCH3), 2.63-2.73 (m, 2H, CH2), 2.23 (m, 1H, CH) 1.8-2.0
(m, 3H, CH2-CH).
Example 7
[0028] A mixture of Compound G as free base (.5 g, 1.94 mmol) and sodium hydride (60%) (0.085
g, 3.54 mmol) was heated at 100 °C under nitrogen until the evolution of H
2 gas ceased. The resultant was dissolved in anhydrous DMF and benzenesulfonylchloride
(0.30 mL, 2.35 mmol) was added drop wise at 0 °C.. The reaction mixture was stirred
at room temperature overnight. Saturated NaHCO
3 solution was added and extracted with CH2Cl
2 (3 x 25 mL). The organic layer was dried over MgSO
4 and the solvent was removed under pressure. The residue was purified by column chromatography
(CH
2Cl
2/MeOH; 9:1) as eluent to give an oil (0.60 g, 76 %) and converted to hydrochloride
salt. The hydrochloride salt was recrystallized from ethanol and anhydrous ether mp
259-261 °C.
Reference
EXAMPLE 8
[0029] 6,7,8,9-tetrahydro-2-methoxy-10-(N,N-dimethylaminoethyl)pyridol[1,2-a]indole-9-one
Oxalate (I). A mixture of 5-methoxy-N,N-dimethyltryptamine (free base) (2.00 g, 9.17mmol)
and 60% NaH (0.41 g, 10.1mmol) was heated at 100 °C under N
2 until evolution of gas had ceased. The resultant mass was dissolved in anhydrous
DMF (25 ml) and anhydrous g-butryolactone 1.4 mL,18.2 mmol) was added in dropwise
manner at room temperature. The reaction mix was heated at reflux for 20 h, cooled
to 0 °C, and acidified by the addition of an ethereal solution of HCl. Additional
Et
2O was added to the resulting suspension and the supernatant was decanted The residue
was dissolved in PPE (52.5 mL) and CHCl
3 (100 mL) and the reaction mixture was heated at reflux for 3 h under N
2. The resulting mixture was neutralized by the addition of 15% NaOH solution at ice-bath
temperature, and extracted with CH
2Cl
2. The organic portion was dried (MgSO
4) and solvent was removed under reduced pressure. The residue was purified by flash
chromatography on silica gel (CH
2Cl
2/MeOH; 20:1) to give 0.52 g (20%) of 6,7,8,9-tetrahydro-2-methoxy-10-(N,N-dimethylaminoethyl)pyridol[1,2-a]indole-9-0ne
free base as a yellow oil.
1H-NMR (DMSO-d
6) □ 7.35 (d, 1H, J=8.79 Hz) 7.18 (s, 1H), 6. 88 (d, 1H, J=8.85 Hz), 4.06 (t, 2H, J=6.60
Hz), 3.80 (s, 3H), 3.42-3.36 (m, 2H), 3.17-3.12 (m, 2H), 2.85 (s, 6H), 2.66-2.62 (m,
2H0, IR (CHCl
3) 1648
m-1. A small sample was converted to the oxalate salt; mp 191-192 °C.
Reference
EXAMPLE 9
[0030] 6,7,8,9-tetrahydro-2-methoxy-10-(N,N-dimethylaminoethyl)pyridol[1,2-a]indole Oxalate
(J). A solution of 1.0 M borane/THF (2 mL, 2mmol) was added ion a dropwise manner
to ice-bath cooled 6,7,8,9-tetrahydro-2-methoxy-10-(N,N-dimethylaminoethyl)pyridol[1,2-a]indole-9-one
Oxalate (290 mg, 1.01 mmol) under N
2. The reaction mixture was allowed to stir at room temperature for 2 h. Acetone (3
ml) was added , and the reaction mixture was heated at reflux for 1 h to quench the
unreacted borane reagent. The solvent was removed under reduced pressure. The residue
was purified by flash chromatography on silica gel (hexane/EtOAc; 4:1) to give 207
mg (75%) of a light yellow oil.
1H-NMR (DMSO-d
6) □ 7.34 (d, 1H, J=8.85 Hz) 7.21 (s, 1H), 4.08 (t, 2H, J=6.65 Hz), 3.79 (s, 3H), 3.40-3.35
(m, 2H), 3.30-3.25 (m, 2H), 3.06-3.01 (m, 2H) 2.83 (s, 6H) 1.76-1.69 (m, 2H), 1.40-1.31
(m, 2H). A small portion was converted to its oxalate salt; mp 114-115 °C.
Reference
EXAMPLE 10
[0031] 1-Benzenesulfonyl-5-methoxy-N,N,-dimethyltryptamine oxalate. A mixture of 5-methoxy-N,N-dimethyltryptamine
(free base) (4.35g, 19.93 mmol) and 60% NaH (0.87 g, 21.75 mmol) was heated at 100
°C under N
2 until evolution of H
2 gas ceased. The resultant mass was dissolved in anhydrous DMF (21 ml) and benzenesulfonyl
chloride (2.8 ml, 21.94 mmol) and 60% NaH (0.87 g, 21.75 mmol) was added in a dropwise
manner at 0°C. The reaction mixture was allowed to stir at room temperature overnight.
Saturated NaHCO
3 solution was added and the mixture was extracted with CH2Cl2. The organic portion
was dried (MgSO
4)and the solvent was removed under reduced pressure. The residue was purified by flash
chromatography on silica gel (CH
2Cl
2/MeOH; 9:1) to give 4.39 g of an oil (61%).
1H-NMR (CDCl
3) □ 7.89-7.87(m, 1H,) 7.83 (d, 2H, J=8.0 Hz), 7.51 (t, 1H, J=7.8 Hz), 7.34 (s, 1H),
6.93-6.92 (m, 2H), 3.82 (s, 3H), 2.80 (t, 2H, J=7.8 Hz) 2.59 (t, 2H, J=7.8 Hz), 2.33
(s, 6H). IR CHCl
3, 1357, 1115 c
m-1. The oxalate salt was prepared and recrystallized from an acetone/Et2O mixture; mp
224-226 °C.
Reference
EXAMPLE 11
[0032] 1-Benzenesulfonyl-5-methoxy-2-n-propyl-N,N-dimethyl tryptamine. A 2.5 M solution
of nBuLi (1.4mL, 3.5 mmol) was added in a dropwise manner to a stirred solution of
1-Benzenesulfonyl-5-methoxy-N,N,-dimethyltryptamine oxalate (free base) (1.00g, 2.79mmol)
in DME (4 mL) at -10 °C under N
2. The resulting solution was allowed to stir for an additional 10 min at -10 °C, and
then nPrl (0.35 mL, 3.59mmll) was added. The reaction mixture was allowed to stir
for 1 h at -10 °C. Saturated NaHCO
3 solution was added and the reaction mixture was extracted with CHCl2. The organic
portion was washed with brine and dried (MgSO
4); the solvent was removed under reduced pressure and the residue was purified by
flash chromatography on silica gel. (CH
2Cl
2/MeOH;30:1). To give 0.19 g ofa bright yellow oil.
1H-NMR (CDCl
3) □ 8.06 (d, 1H, J=8.79 Hz), 7.62 (d, 2H, J=8.22Hz), 7.51-7.46 (m, 1H), 6.89-6.85
(m, 1H), 3.85 (s, 3H), 2.96-2.89 (m, 4H) 2.63-2.57 (m, 2H), 2.48 (s, 6H) 6.87, 1.73
(q, 2H, J=7.51 Hz), 1.00 (t, 3H, J=7.51Hz). IR CHCl
3, 1355 c
m-1.
EXAMPLE 12
5-HT derivative binding to 5-HT6 receptor
[0033] The binding assay employed human 5-HT
6 stably transfected to HEK 293 human embryonic kidney cells with [
3H] lysergic acid diethylamide (70Ci/mmol; Dupont NEN) as radioligand. Radioligand
was diluted in incubation buffer in borosilicate glass vials and protected from light.
Competing agents (1mM stock solution) were dissolved in DMSO or saline and stored
at -20 0 C. Dilutions of compounds were made using incubation buffer in 96-well plates
and mixed by multichannel pipetting. Serial dilutions (1 in 4) started at a final
concentration of 10,000 nM. Final concentrations >10,000 nM were individually prepared
from the 1mM stock solution. Nonspecific binding was defined by 100mM serotonin creatinine
sulfate (Research Biochemicals) prepared fresh in incubation buffer at the time of
each determination, and protected from light. in incubation buffer at the time of
each determination, and protected from light. Reaction volumes were as follows: 200ml
incubation buffer (50 mM tris, 0.5m M EDTA, 10mM MgCl
2), pH 7.4 at 22 0 C,100 ml test agent or serotonin (100mM) or buffer, 100 ml [3H]lysergic
acid diethylamide (2nM final concentration) and100 ml membrane preparation (15 mg
protein). The incubation was initiated by the addition of membrane homogenate and
the plates vortexed. The plates were incubated, with protection from light, by shaking
at 37 0 C for 60 min. The binding reaction was stopped by filtration. The samples
were filtered under vacuum over 96 well glass fiber filters, presoaked in 0.3% PEI
in 50 mM tris buffer (pH 7.4) for at least 1H and then washed 6x with 1 ml of cold
50 mM tris (4 0 C, pH 7.4) using a Packard Filtermate Harvester. The unifilter plates
were dried overnight in a 37 ° C dry incubator. The unifilter bottoms were sealed
and 35 ml of Packard MicroScint was added. The plates were allowed to equilibrate
for 1h and were then sealed using a Packard TopSeal P with the Packard Plate Micromate
496. Plates were counted by liquid scintillation spectrometry. Each well was counted
for 3 min. Compounds were initially assayed at 1000 and 100 nM. If a compound caused
at least 80% inhibition of [
3H]lysergic acid diethylamide binding at 1000 nM, it was further tested and a Ki determined.
The range of concentrations chosen was so that the middle concentration would produce
approximately 50% inhibition.
Table1 shows the affinity for 5-HT
6 receptors of claimed compounds derived from the general formula in formula 2 above.
COMPOUND |
R1 |
R2 |
R3 |
R4 |
R5 |
Ki(nm) |
A |
Me |
Me |
5-OMe |
H |
Me |
60 |
B |
Me |
Me |
5-OMe |
H |
Et |
16 |
C |
Me |
Me |
5-OMe |
H |
nPr |
185 |
D |
Me |
Me |
5-OMe |
H |
Phenyl |
20 |
Table 2 shows the results of affinity for 5-HT6 receptors of daimed and reference
compounds derived from the general formula in formula 3 above.
COMPOUND |
R1 and R2 |
R4 |
Ki(nm) |
E (Reference) |
Me |
-CH2-phenyl |
136 |
F (Reference) |
H |
-Ch2-phenyl |
302 |
G |
Me |
H |
168 |
H (Reference) |
Me |
-SO2-phenyl |
2 |
Table 3 shows the results of affinity for 5-HT
6 receptors of reference compounds derived from the general formula in formula 4 above.
COMPOUND |
X |
Ki(nM) |
I |
O |
84 |
J |
H2 |
1030 |
EXAMPLE 13
Characterization of 5HT6 Selectivity
[0034] Selected compounds were examined to determine their specificity of binding to the
5-HT
6 receptor. Compounds were examined at more than 30 receptor populations. Assays for
the following receptors were performed as per the NIMH Psychoactive Drug Screening
Program. The compounds failed to displace radioligand (i.e., <50% displacement) at
a concentration of 10,000 nM at most receptors. Where more than 50% displacement was
observed, Ki values were determined and the data are reported in the following table.
It can be seen that the compounds are selective for 5-HT
6 receptors.
[0035] Table 4 shows the 5-selectivity of several compounds of the invention.
TABLE 4 Ki, nM(SEM)
Receptor Population |
COMPOUND A |
COMPOUND B |
COMPOUND D |
CONTROL AND AGENT |
NET |
6,380(3190) |
>10,000 |
>10,000 |
Nortriptyline 6.3 (1.2) |
SERT |
>10,000 |
>10,000 |
4,700(1550) |
Fluoxetine 3.5(0.7) |
h5-HT1A |
200(60) |
170(54) |
1,470(310) |
WAY 1000,635 0.6 (1.5) |
h5-HT1D |
250(180) |
290(700) |
6,225(70) |
Ergotamine0. 8 (0.6) |
h5-HT1E |
1,800(600) |
520(180) |
>10,000 |
Serotonin0.5 (.015) |
r5-HT2A |
>10,000 |
>10,000 |
470(10) |
Clozapine 9(1) |
r5-HT2C |
4,020(640) |
1,810(490) |
675(180) |
Clozapine 23(5) |
h5-HT5A |
10,450(2195) |
4,620(650) |
5,160(930) |
Ergotamine 22(3) |
h5-HT7 |
145(34) |
300(60) |
155(35) |
Clozapine 9(2) |
h5-Ht6 |
60(13) |
16(4) |
20(5) |
Clozapine 10(3) |
[0036] Compounds displayed Ki values of>10,000 nM at the following populations of receptors:
histamine, NMDA, PCP, acetylcholine, opiate, and vasopressin receptors. Ki values
were >10,000 nM for compounds A and B at hD1,rD2,rD3,rD4, and hD5 receptors and 10,000
nM for D at hD1,rD2, and rD4 receptors. Compound D produced 70% inhibition at rD3
and hD5 receptors. NET and SERT represent the norepinephrine and serotonin transporters.
Ki values for all three compounds were >10,000 at the dopamine transporter.
EXAMPLE 14
[0037] cAMP activation assays. Human 5-HT
6 receptors stably expressed in 293 HEK cells were grown in 24-well plates to near
confluence and 18 h prior to the assay the medium was replaced with DMEM containing
dialyzed 10% Fetal Calf Serum. For the assay, the medium was aspirated and replaced
with fresh DMEM without serum and incubated with various concentrations of compounds
of the invention in a total volume of 0.5 ml for 15 min. The assay was terminated
by aspiration and the addition of 10% trichloroacetic acid (TCA). The TCA extract
was used for cAMP determinations. (Data represent the mean of N=4 separate determinations).
Results of cAMP activation by various compounds of the invention are shown in the
attached Drawing of Figure 1.